Yongping Fan

Effects of Bu Shen Yi Sui Capsule on Th17/Treg cytokines in C57BL/6 mice with experimental autoimmune encephalomyelitis

BackgroundT helper (Th) 17 and regulatory T (Treg) cells play a critical role in the pathogenesis of multiple sclerosis (MS) disease. Bu Shen Yi Sui Capsule (BSYSC), a traditional Chinese medicine formula, has been used clinically for the treatment of MS patients in China.MethodsTo evaluate the neuroprotective effects and the underlying mechanisms of BSYSC on MS, experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice was induced with myelin oligodendrocyte glycoprotein (MOG) 35–55. Th17 and Treg cells and the related cytokines were detected by flow cytometry, ELISA, real-time quantitative reverse transcription PCR, western blot and immunohistochemistry.ResultsWe found that BSYSC improved neurological function, reduced inflammatory cell infiltration and damage to the axons and myelin in the brain and spinal cord. BSYSC down-regulated markedly the ratio of CD4 + IL-17+/CD4 + CD25 + FoxP3+ T cells in the spleen, decreased the cytokines of IL-17A, IL-6, IL-23, TGF-beta1 in the brain, and dropped the ratio of IL-17A and FoxP3 mRNA and protein in the brain or spinal cord at different stages.ConclusionsThe study demonstrated that BSYSC had a strong neuroprotective effect on EAE mice. The protective mechanisms of BSYSC might be associated with mediating the regulation of Th17/Treg cells.

Impact of Depression, Fatigue and Disability on Quality of Life in Chinese Patients with Multiple Sclerosis

The aim of this work is to investigate and analyze the main factors that influence the quality of life (QOL) of multiple sclerosis (MS) patients. The QOL (multiple sclerosis impact scale), disability (expanded disability status scale), fatigue (modified fatigue impact scale) and depression (Beck Depression Inventory) were assessed in 100 MS patients. Correlation analysis shows that QOL is positively correlated with disability status, fatigue and depression, i.e., the more severe the disability, fatigue and depression, the worse the QOL. Multiple linear regression analysis indicated that Expanded Disability Status Scale grade and fatigue have important predictive value on the somatic QOL of MS patients. On the other hand, depression and fatigue have important predictive value on the mental QOL of MS patients. The QOL of MS patients is influenced by various factors, nursing care that focuses on patient disability, fatigue and depression should be strengthened.

Bushen Yisui Capsule ameliorates axonal injury in experimental autoimmune encephalomyelitis.

A preliminary clinical study by our group demonstrated Bushen Yisui Capsule (formerly called Erhuang Formula) in combination with conventional therapy is an effective prescription for the treatment of multiple sclerosis. However, its effect on axonal injury during early multiple sclerosis remains unclear. In this study, a MOG35-55-immunized C57BL/6 mouse model of experimental autoimmune encephalomyelitis was intragastrically administered Bushen Yisui Capsule. The results showed that Bushen Yisui Capsule effectively improved clinical symptoms and neurological function of experimental autoimmune encephalomyelitis. In addition, amyloid precursor protein expression was down-regulated and microtubule-associated protein 2 was up-regulated. Experimental findings indicate that the disease-preventive mechanism of Bushen Yisui Capsule in experimental autoimmune encephalomyelitis was mediated by amelioration of axonal damage and promotion of regeneration. But the effects of the high-dose Bushen Yisui Capsule group was not better than that of the medium-dose and low-dose Bushen Yisui Capsule group in preventing neurological dysfunction.

Research on the mechanism of Zuogui Pill (左归丸) and Yougui Pill (右归丸) in promoting axonal regeneration in model rats of autoimmune encephalomyelitis

ObjectiveTo study the molecular mechanism of Zuogui Pill (左归丸, ZGP) and Yougui Pill (右归丸, YGP) on axonal regeneration in rats with experimental autoimmune encephalomyelitis (EAE).MethodsEAE rat model was established by bilateral rear pedes subcutaneous injection of antigen made by mixing myelin basic protein (MBP) and complete Freud’s adjuvant (CFA) in the volume ratio of 1:1. The pathological changes of axonal injury and regeneration in the brain and the spinal cord were observed on the 14th (the acute stage) and the 28th day (the remission stage) after modeling, with hematoxylin-eosin (HE) staining, silver stain, and immunohistochemical staining. The rats treated with prednisone acetate were taken as controls.ResultsObservation under the light microscope with HE staining showed a sleeve-like change in rats’ cerebrospinal parenchyma with inflammatory cell infiltration around the small vessels and neuronic denaturation, while silver staining showed excessive tumefaction and abscission of axon, and immunohistochemical analysis showed decreasing of nerve growth factor (NGF) expression at the acute stage of EAE, which was even more remarkable at the remission stage, showing significant difference as compared with the normal control (P<0.05). And the expressions of Nogo A, an axon growth inhibitor, and its receptor (Nogo-66 receptor, Ng R) were significantly higher than those in the normal control at the acute stage (P<0.01). However, after the intervention of ZGP and YGP, the pathological changes and axon damage in rats’ brain and spinal cord were much more alleviated, and the NGF expression was significantly higher than that in the model group at the acute stage (P<0.05). The expression of NGF was even stronger during the remission stage, and a better effect was shown by YGP. As for Nogo A and Ng R expressions, they were significantly lower than those in the model group at the acute stage (P<0.05), but a better effect was shown by ZGP.ConclusionsZGP and YGP can prevent axonal injury and promote the axonal regeneration in rats of EAE, and the possible mechanism is to increase the expression of NGF and reduce the expression of Nogo A and its receptor. However, some differences are observed between the two Chinese preparations in their acting times and points, which provides a certain basis for revealing the modern connotation of the Chinese medicine theory on tonifying Shen (肾)-yin and Shen-yang.

Effect of catalpol on remyelination through experimental autoimmune encephalomyelitis acting to promote Olig1 and Olig2 expressions in mice

BackgroundMultiple sclerosis (MS) as an autoimmune disorder is a common disease occurring in central nervous system (CNS) and the remyelination plays a pivotal role in the alleviating neurological impairment in the MS. Catalpol, an effective component extracted from the Chinese herb Radix Rehmanniae, which has been proved protective in cerebral diseases.MethodsTo determine the protective effects and mechanisms of Catalpol on MS, the mice with experimental autoimmune encephalomyelitis (EAE) were induced by myelin oligodendrocyte glycoprotein (MOG) 35–55, as a model for human MS. Th17 cells were counted by flow cytometric (FCM). The expressions of nerve-glial antigen (NG) 2 and myelin basic protein (MBP) were measured by immunohistochemical staining. Olig1+ and Olig2+/BrdU+ cells were counted by immunofluorescence. Olig1 and Olig2 gene expressions were detected by real-time fluorescent quantitative reverse transcription (qRT) -PCR.ResultsThe results showed that Catalpol improved neurological function, reduced inflammatory cell infiltration and demyelination. It could decrease Th17 cells in the peripheral blood. It increased the protein expressions of NG2 and MBP in mice brains, up-regulated markedly protein and gene expressions of Olig1 and Olig2 in terms of timing, site and targets.ConclusionsThese data demonstrated that Catalpol had a strong neuroprotective effect on EAE mice. Catalpol also plays a role in remyelination by promoting the expressions of Olig1 and Olig2 transcription factors.

Mechanism of Erhuang capsule for treatment of multiple sclerosis.

Erhuang capsule, a typical formula based on traditional Chinese medicine theory, is widely used to ameliorate multiple sclerosis, inflammation and side effects of glucocorticoid treatment. Oligodendrocyte precursor cells are neural stem cells that are important for myelin repair and regeneration. In the present study, Erhuang capsule effectively improved clinical symptoms and neurological function scores, reduced mortality and promoted recovery of neurological functions of mice with experimental autoimmune encephalomyelitis. The mechanism of action involved significant increases in oligodendrocyte precursor cell proliferation in specific regions of the brain and spinal cord, increased oligodendrocyte lineage gene 2 expression and enhanced oligodendrocyte precursor cell differentiation.

Increased plasma levels of epithelial neutrophil-activating peptide 78/CXCL5 during the remission of Neuromyelitis optica

BackgroundIn neuromyelitis optica (NMO), one of the underlying pathogenic mechanisms is the formation of antigen-antibody complexes which can trigger an inflammatory response by inducing the infiltration of neutrophils in lesions. Epithelial neutrophil-activating peptide 78 (ENA 78), known as Chemokine (C-X-C motif) ligand 5 (CXCL5), belongs to the ELR-CXCL family. It recruits and activates neutrophils. The aim of this study was to evaluate ENA 78, IL-1β and TNF-α plasma levels in multiple sclerosis (MS) and neuromyelitis optica (NMO) patients.MethodsENA 78, IL-1β and TNF-α plasma levels were detected in 20 healthy controls (HC), 25 MS and 25 NMO patients using MILLIPLEX® map Human High Sensitivity Cytokine/Chemokine Panels.ResultsPlasma levels of ENA 78 were significantly higher in NMO patients than in HC (P < 0.001) and MS patients (P < 0.05). The NMO patients showed higher plasma levels of IL-1β compared with HC (P < 0.01). Further, increased plasma levels of TNF-α were found in the MS (P < 0.05) and NMO patients (P < 0.001). In addition, NMO patients had higher Expanded Disability Status Scale (EDSS) scores compared with MS patients (P < 0.05). EDSS scores were correlated with plasma levels of ENA 78 in NMO patients (P < 0.05). There were no significant correlations between EDSS scores and plasma levels of ENA 78 in MS patients (P > 0.05).ConclusionsThe overproduction of pro-inflammatory cytokines such as IL-1β and TNF-α during the remission of NMO activates ENA 78, which in turn leads to neutrophil infiltration in lesions. ENA 78 plasma levels were correlated with EDSS scores in NMO patients. Elevated secretion of ENA 78 may be a critical step in neutrophil recruitment during the remission of NMO.

Effect of erhuangfang on cerebral and spinal demyelination and regeneration as well as expression of glial fibrillary acidic protein in rats with experimental allergic ncephalomyelitis

Abstract Background It demonstrates that erhuangfang can improve clinical symptoms of multiple sclerosis and relieve side effects of hormone. However, whether erhuangfang can improve experimental allergic encephalomyelitis (EAE) or not needs a further study. Objective To observe the effect of erhuangfang on neuro-pathology and astrocyte in EAE rats and compare with the effect of hormone. Design Randomized controlled animal study. Settings Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University; College of Traditional Chinese Medicine, Capital Medical University. Materials The experiment was carried out in the Laboratory Center of Capital Medical University from August to October 2005. Ten adult guinea pigs (SPF grade, weighing 400–450 g) and 70 adult Lewis rats (SPF grade, weighing 200–220 g) were selected in this study. Erhuangfang consisted of jiudahuang, shengdi, shuizhi, dabeimu, etc. Methods Experimental intervention: Rats were randomly divided into normal group (n=10), model group (n=20), western medicine group (n=20) and Chinese herb group (n=20). Mixed emulsion, which was consisted of Freunds adjuvant and spinal cord homogenate of guinea pigs, was subcutaneously injected into palms of the two hindfeet of rats in the latter three groups to establish EAE models. Foot pads were injected with saline and then rats were perfused with saline in the normal group. In the model group, models were established as the same as those mentioned above, and rats were also perfused with saline. Rats in the western medicine group were perfused with saline and then 5 mg/kg prednisone acetate suspension. Rats in the Chinese herb group were perfused with erhuangfang decoction (15 g raw materials per kilogram) at 5 days before model establishment. The dosage in the four groups was 3 mL/day per rat. Experimental evaluation: At 28 days after model establishment, rats were randomly selected for cerebral (mainly surrounding cerebral ventricle) and spinal cord (cervical enlargement and lumbar enlargement) collections, and then haematine-eosin (HE) staining and SLG myelin staining were used to observe demyelination and regeneration; meanwhile, immunohistochemical staining was used to observe the expression of glial fibriliary acidic protein (GFAP). Main outcome measures Cerebral and spinal demyelination and regeneration as well as expression of GFAP in EAE rats. Results All 70 Lewis rats were involved in the final analysis. Demyelination and regeneration: Infiltration of inflammatory cells surrounding cerebrum and small venous vessels of spinal cord white matter, demyelination surrounding vessels and plentiful foam cells at myelinolysis sites were observed in the model group. Symptoms were relieved in the western medicine group and the Chinese herb group as compared with those in the model group. While, numbers of inflammatory infiltrated cells and vascular cuffs were decreased in focal region as compared with those in the model group; in addition, areas of softening focus and demyelination were decreased. Expression of GFAP: Volumes and numbers of positive cells of GFAP in white matter region were respectively bigger and higher than those of normal cells in the model group. Plentiful positive cells of GFAP were disorderly aggregated in hippocampus and surrounding small vessel cuffs. While, expression of GFAP was mildly increased surrounding focus in the Chinese herb group; however, GFAP did not express surrounding focus in the western medicine group. In addition, expressions of GFAP were not increased in non-focal region in both Chinese herb group and western medicine group. Conclusion Both erhuangfang and hormone can relieve inflammatory reaction of central nervous system and demyelination of EAE rats. On one hand, erhuangfang can regulate reaction of astrocyte in two ways, relieve reaction and proliferation of astrocyte in non-focal region and maintain the protective effect of astrocyte on brain tissue in focal region; on the other hand, hormone can overall inhibit reaction of astrocyte.

Upregulation of Bcl-2 and Its Promoter Signals in CD4+ T Cells during Neuromyelitis Optica Remission

The homeostatic balance between production and elimination of CD4+ T cells in peripheral blood plays an important role in patients with neuromyelitis optica (NMO). The objective of the present study was to evaluate the anti-apoptosis genes Bcl-2 and its promoter signal (nuclear factor kappa-light-chain-enhancer of activated B cells, NFκB) in CD4+ T cells. Healthy subjects (HS, n = 25) and patients with multiple sclerosis (MS) (n = 25) and NMO (n = 30) in remission were consecutively enrolled in this prospective study between May and December 2015. CD4+ T cells were isolated using magnetic beads coated with anti-CD4 monoclonal antibodies, and gene expression of Bcl-2, NFκB, phosphatidylinositol-4, 5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt), and MAP kinase kinase kinase 7 (MAP3K7) was measured by real-time reverse transcription-polymerase chain reaction (rt-PCR). Cytokines of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were detected using human cytokine multiplex assay. Bcl-2 and NFκB gene expressions were elevated in NMO patients (1.63 ± 0.25; 2.35 ± 0.25) compared with those of HS (0.90 ± 0.11; 1.42 ± 0.22) and/or MS patients (1.03 ± 0.18; 1.55 ± 0.20) (P < 0.05). MAP3K7, but not Akt, was increased in NMO patients (1.23 ± 0.18; 1.56 ± 0.22) (P < 0.01) and was a significant factor related to elevated NFκB gene expressions (P < 0.001). On the other hand, IL-1β and TNF-α were also detected in the study and the results showed that both were elevated in NMO patients (23.84 ± 1.81; 56.40 ± 2.45) (P < 0.01; P < 0.05, respectively). We propose that MAP3K7 induced by IL-1β and TNF-α but not Akt promotes NFκB expression and, in turn, prolongs Bcl-2–mediated survival of CD4+ T cells in NMO patients.

Effect of Bushen Yisui Capsule (补肾益髓胶囊) on oligodendrocyte lineage genes 1 and 2 in mice with experimental autoimmune encephalomyelitis

ObjectiveTo study the effects of Bushen Yisui Capsule (补肾益髓胶囊, BSYSC) on the oligodendrocyte lineage genes (Olig) 1 and Olig2 in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE) in order to explore the remyelination effect of BSYSC.MethodsThe mice were randomly divided into normal control (NC), EAE model (EAE-M), prednisone acetate (PA, 6 mg/kg), BSYSC high-dose (3.02 g/kg) and BSYSC low-dose (1.51 g/kg) groups. The mice were induced by immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55. The neurological function scores were assessed once daily. The pathological changes in mice brains were observed with hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). The protein expressions of myelin basic protein (MBP), Olig1 and Olig2 in brains were measured by immunohistochemistry. The mRNA expressions of Olig1 and Olig 2 was also determined by quantitative real-time polymerase chain reaction.ResultsCompared with the EAE-M mice, (1) the neurological function scores were significantly decreased in BSYSC-treated mice on days 22 to 40 (P<0.01); (2) the inflammatory cells and demyelination in brains were reduced in BSYSC-treated EAE mice; (3) the protein expression of MBP was markedly increased in BSYSC-treated groups on day 18 and 40 respectively (P<0.05 or P<0.01); (4) the protein expression of Olig1 was increased in BSYSC (3.02 g/kg)-treated EAE mice on day 40 (P<0.01). Protein and mRNA expression of Olig2 was increased in BSYSC-treated EAE mice on day 18 and 40 (P<0.01).ConclusionThe effects of BSYSC on reducing demyelination and promoting remyelination might be associated with the increase of Olig1 and Olig2.